Elevated plasma triglyceride levels, a common risk factor for cardiovascular disease, has been attributed to defective metabolism of lipoprotein triglycerides by two enzymes, lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL). LPL requires apolipoprotein C-II (apoC-II) for full activity, whereas HTGL does not have a protein cofactor requirement. It is not clear which step in the overall process at the lipid:water interface is rate-limiting: enzyme binding to the lipoprotein surface; enzyme:activator interaction; substrate binding to the enzyme:activator complex enzyme; acylation, enzyme deacylation; fatty acid desorption from the enzyme; or fatty acid desorption from the interface. Stopped flow kinetic methods have not been used to any extent in studies of these complex systems. Our objective is to apply molecular biological approaches, with emphasis on site specific mutagenesis, to prepare mutant enzymes and apoC-II that differ incrementally in activity. Wild-type and mutant cDNAs will be inserted in eukaryotic and bacterial expression vectors. The enzymes and apoC-II produced in vitro will be used to determine kinetic and thermodynamic parameters for lipid-protein association, protein-protein interaction and catalysis. We plan to identify sequences of LPL and HTGL which constitute (a) the fatty acid binding site, (b) the interfacial or lipid binding site, and (c) for LPL, the apoC-II binding site and dimerization site. Sufficient amounts of the proteins will be purified for rigorous analysis of the enzymatic action and the physiochemical processes that control the rates and specificities of catalysis, using stopped flow methods in particular. The identification of the rate limiting steps in this complex enzymatic reaction will provide molecular details of the mechanism of catalytic action, and the physiochemical regulation of the interfacial reactions.